A vast number of new drug targets are now being identified using a combination of genomics, bioinformatics, genetics, and high-throughput biochemistry. Genomics provides information on the genetic composition and the expression of an organism's genes. Bioinformatics uses computer algorithms to recognize and predict expressional patterns in DNA and structural patterns in polypeptides or proteins, defining families of related genes and proteins. Attempts to evaluate gene expression and to decipher biological processes, including those of disease processes and drug effects, have traditionally focused on genomics; however, genomics cannot provide a complete understanding of the cellular processes that are involved in disease processes and provides little or no information as to, for example, the relative abundance of different proteins in a cell, and the types of post-translational modifications present on proteins.
Proteomics offers a more direct and promising look at the biological functions of a cell and involves the qualitative and quantitative measurement of gene activity by detecting and quantitating expression at the protein level, rather than at the DNA or mRNA level. Proteomics also involves the study of non-genome encoded events including the post-translational modification of proteins, interactions between proteins, and the location of proteins within the cell. The structure, function, or levels of activity of the proteins expressed by a cell are also of interest. Generally, proteomics involves the study of part or all of the status of the total protein contained within or secreted by a cell. As many of the most important cellular processes are regulated by the protein status of the cell, not by the status of gene expression, a need exists to characterize proteins in high numbers in a similar manner as is done in genomics.
The number of chemical compounds available for screening as potential drugs is also growing dramatically due to recent advances in combinatorial chemistry, the production of large numbers of organic compounds through rapid parallel and automated synthesis. The compounds produced in the combinatorial libraries being generated will far outnumber those compounds being prepared by traditional, manual means, natural product extracts, or those in the historical compound files of large pharmaceutical companies. Both the rapid increase of new drug targets and the availability of vast libraries of chemical compounds create an enormous demand for new technologies that improve the screening process.
Current technological approaches for obtaining high-throughput screening of drugs and studying polypeptides or proteins function include multiwell-plate based screening systems, cell-based screening systems, microfluidics-based screening systems (in which interconnected fluid pathways and reaction chambers are engineered to provide a “lab on a chip”), and screening of soluble targets against solid-phase probes, e.g., synthesized drug components. Attachment of the target, rather than the probe, to a solid support in the form of an array is particularly promising and can be used to characterize proteins, protein-protein interactions and enzyme catalysis. These high-throughput screening assays are based on a variety of protein separation techniques followed by identification of the separated proteins. The most popular method for protein separation and identification is based on 2D-gel electrophoresis followed by “in-gel” proteolytic digestion and mass spectroscopy. Alternatively, Edman methods may be used for the sequencing.
The high-throughput method generates massive information on protein and peptide target function as well as the influence of synthetic drug on their function and expression level. In a complex biological system it is known that multiplexed proteins and small molecules are used for signal transduction and eventually are necessary for proper function of the system. Detection of numerous target proteins and polypeptide simultaneously is necessary for understanding biological pathways and diseases in such complex function. In order for the polypeptide or protein arrays to be effective, especially on such a large-scale, specific binding between the target and the probe is required, while non-specific binding has to be minimized.
Surface treatment is a key factor for the success of an assay in a high throughput format by reducing non-specific binding. Non-specific binding, or binding of biomolecules, such as polypeptides, proteins, and DNA, to the surface of the array, is a major problem in the design of surface binding assays and can be responsible for generating high background, a decrease in signal to noise ratio, and a decrease in specificity. In microfluidic devices, for example, non-specific binding can additionally lead to peak tailing during separation and a decrease in sensitivity.
Surface treatment with a blocking agent, such as bovine serum albumin (BSA) or milk proteins, can be used to block surface binding sites and reduce background due to non-specific binding. See, e.g., MacBeath & Schreiber, Science, 289: 1760–1763 (2000); Haab et al., Genome Biology, 2(2):1–13 (2001). However, such surface treatment with a blocking agent has various disadvantages. First, it requires a long incubation time for the surface to be covered with the blocking agent and the final surface has to be carefully stored in order to have fair reproducibility. Although blocking agents are relatively effective, there is a resulting decrease in signal to noise ratio due to the unintentional blocking of the probe molecules and the displacement of the blocking agent with the labeled target. In addition, protein-coated surfaces can obscure the probe molecules, particularly small probe molecules, such as aptamers and low-molecular-weight polypeptides and/or proteins. Although deposition of probe molecules on top of the blocking agent, e.g., BSA, is one possible solution, protein-protein interaction may decrease the binding between the target protein and probe molecule. Non-specific binding can also occur even after treatment with a blocking protein if a labeled protein displaces this blocking protein. For surfaces and channels that are part of microfluidic devices, coating with a non-essential blocking protein is also impractical due to the risk to bleeding, which can lead to a decrease in separation/detection efficiency, a shift in baseline, and contamination of the system.
Surface modification with hydrophilic molecules, such as polyethylene glycol (PEG) and dextran, is another approach that has been utilized to decrease non-specific binding of biomolecules, including proteins. See Piehler et al., Biosensors & Bioelectronics 15: 473–481 (2000); J. Milton Harris, ed., Polyethylene Glycol Chemistry, Plenum Press, New York, 1992; Frazier et al., Biomaterials, 21: 957–966 (2000). To yield a high quality surface resistant to non-specific binding, PEG and other hydrophilic molecules must be applied to a substrate with sufficient density and surface coverage, which has been proven to be a problem in prior approaches.
In one approach taken by Piehler, the surface of a substrate is modified by using glycidyloxipropylsilane to obtain an epoxy-moiety, which is then reacted with a reagent, followed by reaction with PEG. The epoxy moiety serves as an adhesive layer. Such a scheme requires tedious steps that increase the cost of the process and decrease its efficiency, particularly in light of a need for an adhesive layer. Additionally, an epoxy-moiety that serves as the adhesive layer is highly reactive, resulting in additional precautions that must be taken during the manufacturing process. Lee and Laibinis (Biomaterials 19: 1669–1675 (1998)) have taken another approach by coating a substrate with oligo (ethylene glycol)-terminated alkyltrichlorosilanes. This approach requires tedious synthetic steps at highly controlled anhydrous conditions because trichlorosilane is highly reactive and toxic. It also requires the protection of terminal functional groups, such as hydroxyl groups, to limit inter- or intra-molecular reaction with the trichlorosilane moiety. For many applications in which hydroxyl groups are needed for probe attachment through covalent linkage, an extra deprotection step is required. These factors make large-scale production difficult and costly.
Accordingly, there exists a need in the art for preparing an effective biomolecule resistant surface that can be prepared easily in a routine fashion with a minimal number of steps.